The present invention relates to receptors and ligands consisting of human Notch gene products, and uses of these receptors, ligands and derivatives thereof to modulate cell-cell interaction in biological processes and conditions including angiogenesis and cancer.
The Notch pathway is involved in cell fate determination and differentiation, and signaling through the Notch pathway receptors is an evolutionarily conserved mechanism for cell-cell interaction. Notch proteins, of which four have been identified in humans (Notch 1, Notch 2, Notch 3 and Notch 4), are a family of closely related transmembrane receptors. Notch 4 is expressed specifically in endothelial cells (Shiratoshi, Y., Genes Cells 2:213-224, 1997; Uyttendaele, H., Development 122:2251-2259, 1996) and may play an important role in angiogenesis. When Notch is activated by a ligand, its intracellular domain is proteolytically cleaved and transported to the nucleus, along with CSL (CBF-1/Su(H)/Lag-1/RBP-JK) transcription factor to activate transcription of downstream effectors. The resulting effector can repress the transcriptional activity of other genes encoding transcription factors for entry into terminal differentiation. The ligands that interact with the extracellular portion of Notch include Delta, Serrate, and Jagged; the ligands also are transmembrane proteins.
Adjacent cells of identical lineage can follow separate pathways of differentiation as a result of the Notch pathway. Sample pathways of differentiation include axis formation, cartilage formation, and somite formation. Through a process of lateral inhibition, one cell can suppress the neighboring cells from following the same path of differentiation. In one model, a Notch receptor is expressed on the cell surface of a xe2x80x9csuppressedxe2x80x9d cell, and interacts with a Notch ligand located on the cell surface of a dominating cell. After ligand interaction with a Notch receptor, the intracellular domain of the Notch receptor is cleaved and transported to the nucleus, where it forms a complex and affects gene transcription. (Lendahl, U., BioEssays 20:103-107, 1998.)
The ligand itself plays an important role in determining the fate of cells in the vicinity of Notch-expressing cells. In Drosophila, ligands including Delta and Serrate have been identified and studied. The corresponding genes in mammals include Dll-1 (Delta-1) and Dll-3 (Delta-3), as well as Jag-1 and Jag-2. Mutations and decreased expression of Delta are related to phenotypic changes, and a translocation in the human Notch 1 locus (TAN-1) has been found in T-cell acute lymphoblastic leukemia/lymphoma (Ellisen et al., Cell 66:649-661, 1991).
Mutations in the human Jagged 1 gene are associated with Alagille syndrome, which involves abnormal development of liver, heart, skeleton, eye, and face. Alagille patients also exhibit valvular and arterial stenonis and high incidence of intracranial hemorrhage. Four separate mutations, all frameshifts, have been identified in patients with the syndrome. (Li, L. et al., Nature Genetics 16:243-251, 1997; Oda, T. et al., Nature Genetics 16:235-242, 1997.) The mutations are likely to interfere with the ability of Jagged 1 to interact with Notch, thereby affecting the differentiation of cells whose fate would otherwise be determined by interaction of Notch with functional Jagged 1. Mice rendered genetically deficient for Jagged 1 exhibit defects in vascular development (Xue et al., Hum. Mol. Gen. 8:723, 1999). These results are consistent with the hypothesis that Jagged 1 is also involved in vascular development and integrity.
Mutations in Notch 3 are related to a syndrome known as CADASIL, for cerebral autosomal arteriopathy with subcortical infarction and leukoencephalopathy. (Jontel, A. et al., Lancet 350:1511-1515, 1997.) Missense mutations in the extracellular domain were found in 45 out of 50 CADASIL patients in one reported study. (Salloway, S. et al, J. Geriatr. Psychiatry Neurol. 11:71-77, 1998.) CADASIL patients exhibit recurrent ischemic stroke and severe vascular smooth muscle cell defect. Thus, mutations in the Notch gene itself can affect vascular integrity in adults.
In view of the importance of this signaling pathway and its role in human cell differentiation and disease, there is a need in the art for identification of genes involved in the pathway, and for methods and therapeutic agents for intervening in diseases and conditions related to defects in the Notch pathway.
The present invention relates to nucleotide sequences of human Notch ligand genes, and amino acid sequences of the encoded proteins, as well as derivatives and fragments thereof wherein the derivatives and fragments exhibit biological activity such as binding to Notch receptors.
The invention also relates to methods of modulating angiogenesis by using the Notch ligands of the invention, and derivatives and fragments thereof.
The invention further relates to modulation of endothelial cell proliferation using polynucleotides encoding all of part of the Notch ligands of the invention, such as antisense oligonucleotides that can target nucleic acid encoding the Notch ligand.
The invention still further relates to methods of modulating the development and maturation of T-cells and other cells of the immune system, thereby regulating cell-mediated immunity and antibody responses to alleviate conditions such as rheumatoid arthritis.
Notch ligands of the invention include those encoded by the 3md3 gene and the 2hd1 gene.
The invention relates to an isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:
(a) a polynucleotide encoding amino acids from about 1 to about 583 of SEQ ID NO:2;
(b) a polynucleotide encoding amino acids from about 2 to about 583 of SEQ ID NO:2;
(c) a polynucleotide encoding amino acids from about 1 to about 81 of SEQ ID NO:4;
(d) a polynucleotide encoding amino acids from about 2 to about 81 of SEQ ID NO:4;
(e) the polynucleotide complement of the polynucleotide of (a)-(d); and
(f) a polynucleotide at least 90% identical to the polynucleotide of (a)-(e).
The invention also relates to an isolated nucleic acid molecule consisting of a nucleic acid comprising 50-1752 contiguous nucleotides from the coding region of SEQ ID NO:1.
The invention further relates to an isolated nucleic acid molecule comprising a polynucleotide encoding a polypeptide wherein, except for at least one conservative amino acid substitution, said polypeptide has an amino acid sequence selected from the group consisting of:
(a) amino acids from about 1 to about 583 of SEQ ID NO:2;
(b) amino acids from about 2 to about 583 of SEQ ID NO:2;
(c) amino acids from about 1 to about 81 of SEQ ID NO:4; and
(d) amino acids from about 2 to about 81 of SEQ ID NO:4.
The invention still further relates to an isolated polypeptide comprising amino acids at least 95% identical to amino acids selected from the group consisting of:
(a) amino acids from about 1 to about 583 of SEQ ID NO:2;
(b) amino acids from about 2 to about 583 of SEQ ID NO:2;
(c) amino acids from about 1 to about 81 of SEQ ID NO:4; and
(d) amino acids from about 2 to about 81 of SEQ ID NO:4.
The invention also relates to a complex comprising a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
The invention further relates to a complex comprising a fragment of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and a Dishevelled protein wherein said fragment is capable of forming a complex with said Dishevelled protein.